This invention relates to compositions and methods for killing bacteria.
Throughout recorded history virulent bacterial infections have been a bane to mankind. Until recently, it was assumed that drug antibiotics had largely eradicated virulent bacteria. It is now apparent, however, that bacteria have circumvented the effects of single-point targeted drug antibiotics. Consequently, there is a need to develop new anti-bacterial agents that can be used to supplement or replace conventional drug antibiotics.
Like animal cells, bacterial cells are subject to infectious agents that are present in their environment. Viruses known as bacteriophage, or phage, specifically infect bacterial cells. Bacteriophage are the natural enemies of bacteria and, over the course of evolution, have developed proteins which enable them to infect a bacterial host cell, replicate their genetic material, usurp host metabolism, and ultimately kill their bacterial host cell.
Research into the use of bacteriophage as therapeutic agents for treatment of bacterial infection began sometime in the late 19th century, predating the development of conventional drug antibiotics. By 1920, Edward Twort and Felix d""Herelle, two noted pioneers in bacteriophage research, were isolating bacteriophage from several bacterial species and using them as anti-bacterial agents. During the early 1940xe2x80x2s, however, antibiotics were introduced to the world as a broad range treatment for bacterial infections, and bacteriophage therapy research went into decline.
Early clinical studies of phage therapy were plagued with poor experimental design, with few controls and little documentation, variable success due to the indiscriminate use of phage to treat a broad range of bacterial infections, and the use of procedures that introduced bacterial toxins into patients and loss of effectiveness of the isolated phage.
The lack of knowledge and scientific expertise needed to understand bacteriophage and their interaction with bacteria also hindered efforts to improve phage therapy. For example, differences between the biological interaction of bacteriophage strains with their species-specific bacterial host in vitro as compared to in vivo have posed considerable difficulty. Although bacteriophage can be selected for their lytic virulence (immediately replicating and then inducing bacterial host cell lysis following infection) in vitro, such selection does not guarantee against the conversion of a seemingly lytic phage to a temperate phage (entering into a state of lysogeny via integration of the bacteriophage genome into the bacterial genome followed by a quiescent period during which lytic proteins are not expressed) in vivo. These conversions result in lysogenic bacteria that are resistant to further bacteriophage infection, thus reducing the effectiveness of phage therapy.
Since the early 1940xe2x80x2s drug antibiotics have become the choice for treating virulent bacterial infections. Several problems associated with this approach are now becoming evident. The misuse and overuse of drug antibiotics has contributed to the rise of antibiotic resistant bacterial strains. Moreover, since drug antibiotics are non-specific with respect to the types of bacteria that they effect, the bacterial flora that naturally occur within the body are killed along with the disease-causing bacterial pathogen. At least 200 identified bacterial species normally inhabit the human body, and many of the these species synthesize and excrete vitamins vital for human health, promote the development of certain tissues, e.g., lymphatic tissue, e.g., Peyer""s patches, and stimulate the production of cross-reactive xe2x80x9cnaturalxe2x80x9d antibodies that react with pathogenic bacteria. Moreover, natural bacterial flora greatly inhibit colonization by non-indigenous bacteria through normal niche colonization or by producing substances and bacteriocins that can inhibit and kill foreign bacteria. Conventional broad spectrum antibiotics risk killing the non-pathogenic bacteria that are responsible for these beneficial effects.
Bacterial drug resistance was evident at the onset of drug antibiotic therapy, and drug resistant virulent strains of both gram-negative bacteria (including pathogenic strains of Escheria coli) and gram-positive bacteria (including pathogenic strains of Staphylococcus and Streptococcus) have become increasingly resistant to drug antibiotics. This increased resistance arises primarily from selection for virulent-resistance strains by the presence of drug antibiotics, resulting in the lateral transfer of resistance genes between different strains and species of bacteria. Epidemic outbreaks have been attributed to a single clone of a benign or virulent progenitor, as well as spontaneous multi-clonal populations within a community setting when drug antibiotic usage is increased. Although decreased usage of antibiotics may improve the odds of generating a population of virulent bacteria that are less resistance towards antibiotics, much contradictory evidence is beginning to surface. For example, a study in Finland found that the incidence of Streptococcus pyogenes resistance to macrolide decreased after macrolide treatment was reduced in favor of treatment with erythromycin. However, a follow-up study reported a subsequent 17% increase in Streptococcus pyogenes resistance to erythromycin. Another growing concern is the increasing number of multi-resistant bacteria. In 1968 approximately 12,500 people in Guatemala died from an epidemic of Shigella, caused by a bacterial strain that contained a plasmid encoding genes resistant to four different antibiotics (Davies (1996) Nature 383:219). Population genetics studies of virulent bacteria causing disease outbreaks or increases in frequency and virulence have shown that the distinct clones responsible for the acute outbreaks are often characterized by unique combinations of virulence genes or alleles of those genes.
Increasing drug antibiotic resistance has resulted in increased dosage levels and duration of antibiotic treatment. These practices are associated with hypersensitivity and serious side effects in a growing number of patients (see Cunha (2001) Med Clin North Am 85:149; Kirjavainen and Gibson (1999) Ann Med 31:288; Lee et al. (2000) Arch Intern Med 160:2819; and Martinez et al. (1999) Medicine 78:361). The increasing hypersensitivity and side effects are not being seriously addressed and have so far been clinically under-evaluated (Demoly et al. (2000) Bull Acad Natl Med 184:761; and Gruchalla (2000) Allergy Asthma Proc 21:39). As an example of one serious side effect that is becoming increasingly prevalent, especially in children, the use of antibiotics has been shown to be positively associated with the development of asthma and atopy. The mechanisms underlying these associations remain largely unknown (von Hertzen (2000) Ann Med 32:397).
Drug antibiotics and their effects are not isolated to individuals under the supervision of a doctor""s care, but are a communal health issue. Molecular population studies have identified healthy humans that are VRE (vancomycin-resistant enterococci) carriers. An increase in VRE strains in healthy farm animals is associated with the increased use of the antibiotic avoparcin. There is currently a tentative link between the consumption of farm animals and VRE transference to people (Bates (1998) J Hosp Infect 27:89). Data on antibiotic resistance profiles of several food born pathogens provides ample evidence that antibiotic resistance traits have entered the microflora of farm animals and the food supply produced from them (Teuber (1999) Cell Mol Life Sci 56:755).
The present invention is based, at least in part, on the development of intracellular peptide toxins and peptide-like toxins that are toxic to a cell when inside the cell, but relatively non-toxic to the cell when outside the cell. Such peptide toxins and peptide-like toxins are useful in the production of a recombinant bacteriophage that effectively function as a bacteriocide (i.e., a toxin-phage bateriocide) that can provide a viable alternative to conventional drug antibiotics. The toxin-phage bacteriocide (TPB) include bacteriophage that have been genetically engineered to encode a peptide toxin that can be expressed within the bacterial host cell. Within the bacterial host cell, the peptide toxin is active and functions to kill the bacterial host cell. Importantly, the toxin-phage bacteriocide of the invention retains its activity as a bacteriophage, and is therefore capable of completing the lytic phase of its lifecycle. Completion of the lytic phase of its life-cycle results in both the production of additional toxin-phage bacteriocide and host cell lysis.
Accordingly, in one aspect, the invention features a method of producing a toxin-phage bacteriocide. The method includes: (a) identifying a bacteriophage that is capable of infecting a bacterial cell of interest; (b) preparing a recombinant bacteriophage genome via the introduction of a nucleic acid sequence that encodes an intracellular peptide toxin into the genome of the bacteriophage, wherein the nucleic acid sequence that encodes the peptide toxin is operatively linked to a promoter that is active within the bacterial cell of interest; and (c) allowing the formation of a toxin-phage bacteriocide particle that contains the recombinant bacteriophage genome.
In preferred embodiments, the nucleic acid sequence that encodes an intracellular peptide toxin includes the nucleic acid of SEQ ID NO: 1, which encodes the TPB peptide toxin A amino acid sequence (SEQ ID NO: 2). In other embodiments, the nucleic acid sequence that encodes an intracellular peptide toxin encodes a peptide toxin other than the TPB peptide toxin A, e.g., a peptide toxin that is a variant of the amino acid sequence of TPB peptide toxin A, or a peptide toxin that functions analogously to the TPB peptide toxin A. In some embodiments, a variant of the TPB peptide toxin A includes at least one mutation, e.g., an insertion, deletion, or point mutation. In preferred embodiments, the mutation is located at one or more of amino acids 16, 17, 18, 19, 20, 21, and 22 of SEQ ID NO: 2. In other preferred embodiments, the mutation is a conservative amino acid substitution. In still other preferred embodiments, the mutation does not change the net ionic charge of the resulting TPB peptide toxin variant, as compared to TPB peptide toxin A, under conditions of physiological pH. The following amino acid substitutions are among those considered conservative:
The invention also features a nucleic acid molecule, e.g., an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide consisting essentially of SEQ ID NO: 2. The invention also includes nucleic acid molecules encoding polypeptides comprising or consisting of SEQ ID NO: 2.
Preferably, the nucleic acid molecule encoding the TPB peptide toxin includes a bacterial promoter and other sequences required to direct transcription and translation of TPB peptide toxin in the bacterial cell being targeted. Those skilled in the art can readily obtain promoter sequences and other sequences required for expression.
In preferred embodiments, homologous recombination is used to introduce the nucleic acid sequence that encodes the intracellular peptide toxin into the bacteriophage genome. In related embodiments, homologous recombination is carried out in vitro. In other related embodiments, homologous recombination is carried out in vivo. In other embodiments, the recombinant bacteriophage genome is packaged into bacteriophage particles in vitro or in vivo, thereby resulting in the production of toxin-phage bacteriocide particles.
In a related aspect, the invention features compositions that include at least one toxin-phage bacteriocide. In preferred embodiments, the toxin phage bacteriocide includes a nucleic acid sequence encoding an intracellular peptide toxin. In particularly preferred embodiments, the toxin phage bacteriocide includes a nucleic acid sequence encoding the TPB peptide toxin A (SEQ ID NO: 2). In other embodiments, the toxin phage bacteriocide includes a nucleic acid sequence encoding TPB peptide toxin A variants.
In preferred embodiments, the compositions include a single strain or multiple variant strains of toxin-phage bacteriocide that has been substantially purified away from the bacterial host cells used to produce or amplify the toxin-phage bacteriocide. In other preferred embodiments, the compositions include a toxin-phage bacteriocide that has been substantially purified away from the bacterial host cell medium in which the bacterial host cells were grown during the production or amplification of the toxin-phage bacteriocide. In other embodiments, the compositions include a toxin-phage bacteriocide that has been partially purified from the bacterial host cells and bacterial host cell medium used to produce or amplify the toxin-phage bacteriocide.
In another aspect, the invention features a method of using a toxin-phage bacteriocide to kill a bacterial cell. The method involves contacting bacterial cells (e.g., bacterial cells that include one ore more strains or species of bacteria) with a toxin-phage bacteriocide, such that at least one toxin-phage is able to bind to and infect at least one bacterial cell, and then allowing the toxin-phage that have infected bacterial cells to kill the bacterial cells. In preferred embodiments, the toxin-phage binds to and infects bacterial cells that are of a selected type. In other preferred embodiments, the toxin-phage does not bind to or infect bacterial cells that are not of the selected type. The contacting can occur within a patient, e.g., a human or animal patient, or in vitro. In vitro studies using the gram negative Escheria coli and gram positive Bacillus subtilus have found a 100% non-infectivity in the presence of a foreign toxin-phage.
In some embodiments, an infected bacterial cell is killed as a result of the toxin-phage entering into the lytic phase of its life-cycle, such that the bacterial cell is killed by lysis. In other embodiments, the infected bacterial cell is killed as a result of the expression of the toxic peptide encoded by the nucleic acid molecule that was introduced into the genome of the toxin-phage. In still other embodiments, the bacterial cell is killed by a combination of the toxin-phage entering into the lytic phase of its life-cycle and the expression of the toxic peptide encoded by the nucleic acid molecule that was introduced into the bacterial cell by the toxin-phage. In other embodiments, a bacterial cell that is killed is either a gram-negative or a gram-positive bacterial cell.
In another aspect, the invention features a pharmaceutical composition that includes at least one toxin-phage bacteriocide and at least one pharmaceutically acceptable carrier. In preferred embodiments, the pharmaceutical composition can be used in vivo, e.g., the pharmaceutical composition can be administered, e.g., by parenteral injection or orally, to a subject, to treat a bacterial infection present in the subject. In other embodiments, the pharmaceutical composition can be used topically to treat a bacterial infection present in or on a subject.
In another aspect, the invention features a method of using a toxin-phage bacteriocide to treat a bacterial infection present in or on a subject. In some embodiments, the subject is a farm animal, e.g., a chicken, pig, goat, sheep, cow, or horse. In other embodiments the subject is a plant, e.g., an agricultural product or orchard tree. In other embodiments, the subject is a pet, e.g., a fish, bird, cat, or dog. In still other embodiments, the subject is a mammal, a primate, or a human. In preferred embodiments, the toxin-phage bactericide kills the bacteria that are the cause of the infection. In other embodiments, the toxin-phage bactericide slows or brings to a halt the spread of the bacterial infection. In preferred embodiments, the toxin-phage bactericide helps eliminate the bacterial infection. In other preferred embodiments, the toxin-phage bactericide does not kill the bacterial cells that are not the cause of the infection, e.g., bacterial cells that are normally present in the subject or are beneficial to the subject. In other embodiments, the infection constitutes a localized disease, e.g., a disease of the skin, nervous system, cardiovascular system, respiratory system, digestive system, and urinary and reproductive systems.
In another aspect, the invention features a method of using a toxin-phage bacteriocide to prophylactically treat a potential bacterial infection in a subject. In some embodiments, the subject is a farm animal, e.g., a chicken, pig, goat, sheep, cow, or horse. In other embodiments the subject is a plant, e.g., an agricultural product or orchard tree. In other embodiments, the subject is a pet, e.g., a fish, bird, cat, or dog. In still other embodiments, the subject is a mammal, a primate, or a human. In preferred embodiments, the toxin-phage bactericide kills the bacteria that are the potential cause of infection. In other embodiments, the toxin-phage slows or brings to a halt the growth of the bacteria that are the potential cause of infection. In other preferred embodiments, the toxin-phage bactericide does not kill bacterial cells that are not the potential cause of infection, e.g., bacterial cells that are normally present in the subject or are beneficial to the subject. In other embodiments, the potential bacterial infection can result in acne, e.g., skin acne in a human. In other embodiments, the subject has an injury, e.g., a cut that breaks the outer dermal layer of the skin, an animal bite, a dermal burn, or a surgical wound or incision, or a surgically inserted device, e.g., a catheter, that is highly susceptible to bacterial infection. In still other embodiments, the potential bacterial infection can involve exposure to biological weapons, e.g., anthrax, plague, or tularemia.
In another aspect, the invention features a method of treating an aqueous solution with a toxin-phage bacteriocide such that bacteria present in the solution are killed. In one embodiment, the resulting aqueous solution is partially sterilized and can subsequently be consumed by an animal, e.g., a farm animal, pet, mammal, primate, or human. Treatment of the aqueous solution will reduce the chance of bacterial infection resulting from consumption of the solution. In another embodiment, the aqueous solution is a solution that is subject to bacterial contamination, e.g., the water in a fish tank or wastewater, e.g., sewage.
In another aspect, the invention features a method of treating a surface with one or more toxin-phage bacteriocides such that bacteria attached to the surface are killed or their growth is inhibited. In one embodiment, the surface is part of a device, e.g., a device that is used in medicine (e.g., surgical instruments), agriculture, industrial processes, or water and wastewater treatment. In another embodiment, the surface is covered with a biofilm. In other embodiments, the surface is treated regularly with a toxin-phage bacteriocide such that the formation of a biofilm is prevented or slowed.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
The recombinant toxin-phage bacteriocide (TPB) of the invention is a genetically modified bacteriophage that has been modified to harbor a nucleotide sequence encoding a specialized intracellular peptide toxin. This peptide toxin, e.g., the TPB peptide toxin A, is toxic to cells, e.g., bacterial cells when it is present inside the cell, but not when it is outside of a cell. The TPB allows efficient production of a peptide toxin within cells, thus killing the cells. The TPB of the invention are capable of killing a targeted species of bacteria during both lytic and lysogenic infection. This is in contrast to many therapeutic bacteriophages used previously, which can kill host bacteria only during the lytic phase. The TPB of the invention are species-specific. Therefore, significant numbers of commensal bacterial within the host will not become infected or killed by the TPB. Upon infection the TPB delivers its chromosomal DNA into the bacterial host cell. Lytic toxin-phage reproduction results in additional TPB that burst from the cell and infect additional bacterial host cells. Alternatively, depending on various environmental factors, some TPB infected bacterial cells enter lysogeny, incorporating the TPB chromosomal DNA into their own chromosomal DNA. Upon lysogenization of the bacterial cell, but not limited to this temporal event, the bacterial cell""s transcriptional and translational apparatus produces the intracellular peptide toxin. The intracellular peptide toxin, when presented to a cell internally, kills the cell. Upon death of the cell, the intracellular peptide toxin is released into the extracellular environment. However, intracellular peptide toxins are not significantly toxic to cells when presented externally. For example, TPB peptide toxin A had no observable effect on cultures of E. coli or Bacillus Subtilis growing at 37xc2x0 C. even when present at concentrations as high as 34.6 mM over a 25 hour period. Similarly, TPB peptide toxin A added to cultures of Pichia pastoris yeast cells had no observable effect. Finally, 10 xcexcm TPB peptide toxin A had no observable effect on confluent mouse mammary carcinoma cells growing in EMT6 medium or Hanks Balanced Salt Solution over a 6 hour period.
TPB can be designed to be specific for any selected strain of bacteria, thus desirable bacteria can be spared. Bacteriophage specific for a single bacterial host in nature have been found to remain within the host for as long as the bacterial host specific for that phage is present. Weber-Dabrowska, et al. (1987), Arch Immunol Ther Exp (Warsz) 35(5):563-8, tested for absorption of orally administered anti-staphylococcal and anti-pseudomomas phage in both urine and serum samples of patients with suppurative bacterial infections. No phage was present in any of the 56 patients prior to phage therapy. By day 10, 84% of the serum samples and 35% of urine samples contained phage, indicating bioavailability. The healthy control group exhibited a phage titer drop 100-fold between days 0-5. A comprehensive review of phage therapy (Alisky et al. (1998), J of infection 36:5) concluded that all studies with both human and animals showed no measurable antiphage antibodies generated.
Without being bound by any particular theory, it appears that the TPB peptide toxin A, produced by a TPB of the invention, becomes introduced into internally available membranes of the cell. This has been observed to occur in both bacterial and yeast cells. In vitro studies using a lipid bilayer membrane model suggest that the toxin peptide permeabilizes membranes. Significantly, the TPB peptide toxin A does not appear to harm either bacterial cells or eukaryotic cells when applied externally, e.g., when introduced in a culture of growing cells.
The TPB peptide toxin A of the invention has also been found to be toxic to eukaryotic cell when presented internally. Thus, intracellular peptide toxins can be used to selectively target undesirable eukaryotic cells, e.g., cancer cells or virally infected cells, by selectively delivering the peptide toxins to the interior of the undesirable cells. Thus, the peptide toxins can be targeted to such cells in various ways, e.g., through receptor mediated targeting.
This invention is further illustrated by the following examples that should not be construed as limiting.